The outer membrane of Escherichia coli contains proteins which serve as receptors for certain nutrients, colicins and phages. The role of these receptors in the transport of these ligands, and the interaction of the receptor with other membrane components, is the major target of this research proposal. Our primary focus is the system for the uptake of vitamin B12. The first step in its uptake is its binding to the bfe-coded receptor which also functions for the binding of the E colicins and phase BF23. A subsequent step of uptake is dependent on the tonB gene product, which is also necessary for the uptake of iron chelates and the Group B colicins. The four major aspects of this project include the following. 1) Expression of outer membrane proteins: measurement of the expression of the function of certain of the receptor proteins on the cell surface following cessation of their synthesis while allowing continued cell growth. Also, the regulation of the synthesis of many of these proteins will be studied by a genetic approach, including the isolation of mutants constitutive for their expression. 2) Genetics of B12 uptake: further isolation and characterization of mutations affecting the receptor's substrate binding site(s) and the sites of interaction with other uptake components. 3) Colicin tolerance: study of the action of colicins and the effect of mutations to colicin tolerance in membrane vesicle systems; also, study of interactions between different uptake systems. 4) Properties and identification of the tonB product: first, the characterization of the functional stability of this product and the nature of its turnover, with emphasis on the effect of membrane growth on the insertion and function of this protein. Secondly, attempts will be made to amplify, identify and isolate this protein and determine its location in the cell. All of these studies should provide information concerning the mechanism of these outer membrane-dependent systems.